The invention relates to DNA sequences which code protease inhibitors designated eglins, hybrid vectors containing such DNA sequences, hosts transformed by such hybrid vectors, novel potypeptides which have protease inhibitor activity and have been produced by such transformed hosts, processes for the preparation of these DNA sequences, hybrid vectors and transformed hosts, and processes for the preparation of eglins with the aid of the transformed microorganisms.
Two protease inhibitors which are isolated from leeches (Hirudo medicinalis) and which are designated eglin B and eglin C are known from German Offenlegungsschrift 2,808,396. These polypeptides each consist of 70 aminoacids, have a molecular weight of about 8,100 and are potent inhibitors for chymotrypsin, subtilisin, the animal and human granulocyte proteases elastase and cathepsin G and the mast cell protease chymase (1). Trypsin-like proteases are inhibited to a lesser degree.
Eglin C has the following primary structure (2):
ThrGluPheGlySerGluLeuLysSerPheProGluValValGlyLysThrVal AspGlnAlaArgGluTyrPheThrLeuHisTyrProGlnTyrAspValTyrPheLeuProGluGly SerProValThrLeuAspLeuArgTyrAsnArgValArgValPheTyrAsnProGlyThrAsnVal ValAsnHisValProHisValGly
In contrast to most of the known proteinase inhibitors, eglin C contains no disulfide bridge and, even for a miniprotein, it proves to be unusually stable towards denaturation by acid, alkali or heat and towards proteolytic degradation. The primary structure of eglin B differs from that of eglin C by replacement of the aminoacid 35, tyrosine, by histidine.
The eglins belong to the most potent inhibitors known at present for human and animal granulocyte elastase, and for human granulocyte cathepsin G and bacterial proteases of the subtilisin type. Uncontrolled or excessive release of these cellular proteases in the organism can intensify an inflammation process and cause tissue degradation by non-specific proteolysis. This is particularly due to the fact that these enzymes, which are responsible for intracellular digestion, have an optimum action in the physiological (neutral to weakly alkaline) medium and are capable of rapidly destroying and inactivating natural tissue substances (for example elastin) and humoral factors (for example blood coagulation factors and complement factors). On the basis of their properties known so far, the eglins are therefore of great interest for use in medical therapy (antiinflammation, antiphlogistics, septic shock, pulmonary emphysema, mucoviscidosis and the like).
Only very small amounts of eglins are formed in leeches (about 16 xcexcg/leech). Isolation and purification of the eglins from leeches is therefore very time-consuming and expensive and cannot be carried out on a commercial scale.
On the basis of the enormous advances in so-called recombinant DNA technology (or genetic engineering), it has recently become possible to prepare the most diverse physiologically active polypeptides using this technology.
The present invention is based on the object of providing, with the aid of genetic engineering means, expression systems which allow the microbial preparation of eglins on an industrial scale. In the present invention, this object is achieved by providing hybrid vectors containing a DNA sequence which codes an eglin and which is regulated by an expression control sequence such that an eglin is expressed in a host transformed by these hybrid vectors.
Preparation of DNA Sequences which Code an Eglin
The invention relates to DNA sequences which code an eglin, for example eglin B and, in particular, eglin C, or a modified eglin, for example modified eglin B or, in particular, modified eglin C, the modification consisting of a shortening of the primary structure of the eglin whilst maintaining the eglin activity, and fragments thereof.
Unless defined more specifically, the general designation xe2x80x9ceglinsxe2x80x9d in the context of the present invention is to be understood as meaning polypeptides with proteinase inhibitor activity, the primary structure of which largely corresponds to the primary structures of eglin B or C (structure homology in general up to 80%), but which can also be modified N-terminally, for example Nxcex1-acetylated, Nxcex1-methionylated or Nxcex1-acetylmethionylated on the threonine.
In the case of modified eglins, the modification preferably consists of a shortening of the primary structure of the natural eglins, for example by 1 to 10, in particular 1 to 6, aminoacid units at the N-terminus and/or by 1 to 6, in particular 2, aminoacid units at the C-terminus, derivatives modified on the N-terminus, for example acetylated and methionylated or N-acetylmethionylated derivatives, also being included here.
The invention furthermore relates to processes for the preparation of DNA sequences which code an eglin, for example eglin B and, in particular, eglin C, or a modified eglin, for example modified eglin B or, in particular, modified eglin C, and of fragments thereof, which comprises isolating the eglin structure geno from genomic leech-DNA, or preparing a complementary double-stranded eglin-DNA (eglin-ds cDNA) from eglin-mRNA, and, for the preparation of DNA sequences which code a modified eglin, treating the genomic eglin structure gene or the eglin-ds cDNA with suitable nucleases, or which comprises preparing a corresponding (modified) eglin structure gene or fragments thereof by means of chemical and enzymatic processes.
Genomic eglin-DNA and eglin-ds cDNA are obtained, for example, by methods which are known per se. Thus, genomic eglin-DNA is obtained, for example, from a leech gene bank containing the eglin gene, by cloning the leech-DNA fragments in a microorganism and identifying clones containing the eglin-DNA, for example by colony hybridisation using a radioactively labelled eglin-DNA-specific oligodeoxynucleotide containing at least 15, preferably 15 to 30, deoxynucleotides. The DNA fragments thus obtained as a rule contain, in addition to the eglin gene, further undesired DNA constituents, which can be detached by treatment with suitable exo- or endonucleases.
Double-stranded eglin-cDNA can be prepared, for example, by obtaining mRNA from suitable leech cells, preferably those which have been induced into eglin formation, enriching the eglin-mRNA in the resulting mRNA mixture in a manner which is known per se, using the mRNA as a template for the preparation of single-stranded cDNA, synthesising the ds cDNA therefrom with the aid of an RNA-dependent DNA-polymerase and cloning this in a suitable vector. Clones containing the eglin-cDNA are identified, for example, as described above, by colony hybridisation using a radioactively labelled eglin-DNA-specific oligodeoxynucleotide.
To prepare DNA sequences which code modified eglins, the genomic eglin-DNA or eglin-cDNA obtainable can be treated with suitable exo- and/or endo-nucteases which detach the DNA sections coding the N- or C-terminal eglin aminoacids.
The genomic eglin-DNA obtained in this manner or the eglin-cDNA are preferably linked on the 5xe2x80x2- and on the 3xe2x80x2-end with chemically synthesised adapter oligodeoxynucleotides which contain the recognition sequence for one or more restriction endonuclease(s) and thus facilitate the incorporation into suitable vectors. In addition, the adapter molecule for the 5xe2x80x2-end of the eglin-DNA or -cDNA must also contain the translation start signat (ATG). The translation start signal must be located such that it is followed directly by the codon for the first aminoacid of the eglin.
Since the structure of the natural eglin gene is unknown and the chemical synthesis of an eglin gene offers advantages, especially in respect of time, on the basis of modern synthesis possibilities, chemical synthesis is a preferred embodiment of the present invention.
Chemical Synthesis of an Eglin Gene
The invention particularly relates to a process for the preparation of a structure gene for an eglin or for a modified eglin or of fragments thereof, which comprises chemically synthesising segments of the coding and complementary strand of an eglin gene or modified eglin gene and enzymatically converting the segments obtainable into a structure gene of the eglin or the modified eglin or into fragments thereof.
The invention furthermore relates to double-stranded DNAs which code eglins, for example eglin B or eglin C, modified eglins, for example modified eglin B or modified eglin C, or fragments thereof.
In addition to the codons for the eglins or modified eglins, the DNAs according to the invention contain translation start signals and translation stop signals which make expression in suitable host cells, for example in E. coli, possible, and furthermore nucleotide sequences at the ends which are suitable for incorporation into a vector.
In a preferred embodiment of the invention, the DNA comprises, at the 5xe2x80x2-end, a nucleotide sequence which can be cleaved by a restriction enzyme, followed by the translation start signal, codons for an eglin or for a modified eglin, which, if appropriate, make possible cleaving by a restriction enzyme at one or more sites, a translation stop signal and, at the 3xe2x80x2-end, a nucleotide sequence which can be cleaved by a restriction enzyme. Examples of restriction enzymes which can be used according to the invention are EcoRI, BamHI, HpaII, PstI, AvaI and HindIII.
The invention particularly relates to an eglin-coding, double-stranded DNA consisting of a nucleotide sequence of the formula I and the complementary nucleotide sequence
in which the nucleotide sequence is shown starting with the 5xe2x80x2-end and, for better understanding, the amino acids coded by each triplet are given, and in which D is a direct bond or a nucleotide sequence which codes N-terminal aminoacids of the eglin, and B is a direct bond or the corresponding N-terminal aminoacids chosen from the group comprising
and Dxe2x80x2 is a direct bond or a nucleotide sequence which codes C-terminal aminoacids of the eglin, and Bxe2x80x2 is a direct bond or the corresponding C-terminal aminoacids chosen from the group comprising
and in which A is deoxyadenosyl, T is thymidyl, G is deoxyguanosyl, C is deoxycytidyl, X is A, T, C or G, Y is T or C, Z is A, T, C or G, if Y=C, or Z is A or G, if Y=T, Q is T or A, R is C, and S is A, T, C or G, if Q=T, or R is G and S is T or C, if Q=A, M is A or G, L is A or C, N is A or G, if L=A, or N is A, T, C or G, if L=C, K is A or G, if M=A, or K is A, if M=G, W is Tyr or His, and (X)n and (X)m are each any nucleotide sequences with n and m greater than 3 and less than 100, in particular greater than 5 and less than 12, which can be recognised and cleaved by a restriction enzyme, and fragments of such a double-stranded DNA of the formula I.
The invention particularly relates to an eglin-coding double-stranded DNA of the formula I in which D is a nucleotide sequence selected from the group comprising YTZ AAM QRS TTY, QRS GAM YTZ AAM QRS TTY and ACX GAM TTY GGX QRS GAM YTZ AAM QRS TTY, and Dxe2x80x2 is the nucleotide sequence of the formula CAY GTX CCX CAY GTX GGX, and the other symbols are as defined under formula I.
The invention especially relates to an eglin-coding double-stranded DNA of the formula I, in which D is the nucleotide sequence ACX GAM TTY GGX QRS GAM YTZ AAM QRS TTY and Dxe2x80x2 is the nucleotide sequence CAY GTX CCX CAY GTX GGX, and the remaining symbols are as defined under formula I.
In a preferred embodiment, the DNA sequence contains, at the 5xe2x80x2-end, a nucleotide sequence which can be cleaved by EcoRI, and, in the middle, a nucleotide sequence which can be cleaved by HpaII, and, at the 3xe2x80x2-end, a nucleotide sequence which can be cleaved by BamHI.
The invention especially relates to a double-stranded DNA containing triplets which are preferred by E. coli and which code the aminoacids of eglins or modified eglins. Such triplets are: for glycine (Gly): GGT; alanine (Ala): GCT; valine (Val): GTT; leucine (Leu): CTG; serine (Ser): TCT; threonine (Thr): ACT; phenylalanine (Phe): TTC; tyrosine (Tyr): TAC; Methionine (Met): ATG; asparaginic acid (Asp): GAC; glutamic acid (Glu): GAA; lysine (Lys): AAA; arginine (Arg): CGT; histidine (His): CAT; proline (Pro): CCG; glutamine (Gln): CAG; and asparagine (Asn): AAC.
In the present invention, the codon TTT is also used for phenylalanine and CCA or CCT is used for proline, so that, besides the cleavage site for EcoRI at the 5xe2x80x2-end and for BamHI at the 3xe2x80x2-end and a cleavage site for HpaII, no other cleavage sites are present for the restriction enzymes mentioned. The preferred stop signal (NON) is the codon TAG.
A preferred embodiment of a gene for eglin C in the manner shown above is the DNA of the formula IIa
a preferred embodiment of a gene for eglin B is the DNA of the formula IIb
and preferred embodiments of genes for modified (N-terminally shortened) eglin C polypeptides are the DNAs of the formulae IIc and IId
in which A, T, G and C are as defined under formula I and, for better understanding, the aminoacids coded by each triplet and the cleavage sites for the restriction enzymes are given.
The invention furthermore relates to double-stranded DNA fragments of eglin genes, the ends of which can be cleaved by restriction enzymes, and which can be brought together to form complete eglin or modified eglin genes. Such double-stranded DNA fragments of eglin genes have, in particular, 30 to 70 base pairs.
The invention relates, for example, to the DNA fragments of the formula IIIa [F1(C)], the DNA of the formula IIIaxe2x80x2 [F1(Cxe2x80x2)], the DNA of the formula IIIaxe2x80x3 [F1(Cxe2x80x3)], the DNA of the formula IIIb [F1(B)] and the DNA of the formula IV (F2):
The invention also relates to single-stranded DNA fragments of eglin and modified eglin genes, in particular those which can be joined together by chemical and/or enzymatic methods to give eglin or modified eglin genes. The invention particularly relates to single-stranded DNA fragments with more than twenty nucleotides, in particuLar with 20 to 70 nucleotides.
The invention above all relates to the single-stranded and double-stranded DNA fragments described in the examples.
Methods for the synthesis of DNA have been presented in summary form by S. A. Narang (11). The known synthesis techniques allow the preparation of polynucleotides towards 20 bases in length, in good yield, high purity and in a relatively short time. Suitably protected nucleotides are linked with one another by the phosphodiester method (12), or the even more efficient phosphotriester method (13) or phosphite triester method (14). Simplification of the synthesis of the oligonucleotides and polynucleotides is made possible by the solid phase method, in which the nucleotide chains are bound to a suitable polymer. Itakura et at. (15) use trinucleotides linked by the phosphotriester method in the solid phase synthesis, instead of individual nucleotides, and these can thus be condensed, in a short time and with good yields, for example, to give a polynucleotide with 31 bases. The actual double-stranded DNA can be built up enzymatically from chemically prepared short segments. For this, Khorana et at. (16) use overlapping polynucleotide sequences from both DNA strands, which are held together in the correct arrangement by base-pairing and are then chemically linked by the enzyme DNA-ligase. Another possibility comprises incubating in each case one polynucleotide sequence from the two DNA strands with a short overlapping segment in the presence of the four required deoxynucleoside triphosphates with a DNA-polymerase, for example DNA-polymerase I, a Klenow fragment of polymerase I or T4 DNA-polymerase, or with AMV (avian myeloblastosis virus) reverse transcriptase. The two polynucleotide sequences are thereby held together in the correct arrangement by base-pairing and are supplemented with the required nucleotides by the enzyme to give a complete double-stranded DNA (17). Itakura et al. (18) describe how, on the basis of this principle, a segment 132 base pairs long of the human leucocyte interferon xcex12-gene can be built up in the presence of DNA-polymerase I (Klenow fragment) from 4 chemically synthesised fragments 39 to 42 bases in length, a 40% saving in chemical synthesis in comparison with the method which uses only ligase being achieved.
The present invention particularly relates to a process for the preparation of DNAs which code eglins or modified eglins which are suitable for expression in host cells and the ends of which enable incorporation into vectors, and of fragments thereof, which comprises a) bonding a suitably protected deoxynucleoside to a solid carrier, b) preparing suitably protected di-, tri- or tetra-nucleotides by the phosphotriester or phosphite method, c) linking a deoxynucleoside or oligodeoxynucleotide bound to the carrier with suitably protected mononucleotides or di-, tri- or tetra-nucleotides (the latter prepared according to b)) by the phosphotriester or phosphite method, d) detaching carrier-bound oligodeoxynucleotides between about 20 and about 70 bases in length obtainable according to c) from the carrier, if appropriate purifying them, freeing them from protective groups and phosphorylating the free 5xe2x80x2-terminal hydroxyl groups, el) fusing 2 oligodeoxynucleotides each of about 20 to about 70 bases in length from the coding and the complementary strand and with at least 3, preferably 8 to 15, overlapping base pairs and supplementing them with a DNA-polymerase in the presence of the four deoxynucleoside triphosphates to give double-stranded DNA segments (fragments of the eglin or modified eglin gene), and, if appropriate, linking 2 double-stranded DNA segments with suitable ends phosphorylated according to d), with a ligase to give the structure gene of the eglin or of the modified eglin, or subcloning into suitable vectors 2 obtainable double-stranded DNA segments, and then phosphorytating according to d) and linking with a ligase to give the structure gene of the eglin or modified eglin, or e2) alternatively fusing in each case 2 oligodeoxynucleotides from the coding and complementary strand of, for example, 20 to 70 bases in length and with in each case at least 3, preferably 8 to 15, overlapping base pairs, making up with a DNA polymerase in the presence of the four deoxynucleoside triphosphates and linking with ligase to give the structure gene of the eglin or the modified eglin.
The process according to the invention is known per se, but makes the preparation of eglin-coding DNAs possible only by suitable combination of the conditions and improvements essential to the invention.
A large number of solid carrier materials, such as polystyrene crosslinked in various ways and with various swelling capacities, polyacrylamides, polyacrylamide copolymers polyamides absorbed onto inorganic material, such as kiesilguhr, silica get or alox, or functionalised silanes, can be used in step a). In a preferred embodiment of the invention, crosslinked polystyrenes which are linked via xe2x80x9cspacersxe2x80x9d, such as alkylene groups with 2 to 12 C atoms interrupted by 1 to 5 polar divalent functional groups, such as imino, oxo, thio, oxocarbonyl or amidocarbonyl, with the 5xe2x80x2-OH group of suitably protected deoxynucleosides in a manner which is known per se are used as the solid carrier materials. The reaction of nucleosides of the formula V which are protected in the 5xe2x80x2-position and, if appropriate, in the base part and in which R1 is a protective group which can be detached by acid, such as a triarylmethyl protective group, for example a 4-methoxytrityl or 4,4xe2x80x2-dimethoxytrityl group, or a tri-lower alkyl-silyl protective group, for example a tert.-butyldimethylsilyl group, and in which B is a protected or unprotected base chosen from thymyl, cytosyl, adenyl or guanyl, with succinic anhydride, in the presence or absence of bases, such as pyridine, triethylamine or dimethylaminopyridine, followed by reaction with aminomethylated polystyrene, crosslinked by 0.5 to 2% of divinylbenzene, with the aid of reagents which activate the carboxylic acid radical, preferably N-hydroxysuccinimide, or p-nitrophenol and dehydrating agents, such as carbodiimides, for example dicyclohexylcarbodiimide, is particularly preferred (equation 1).
The reaction is carried out in an inert, non-protic solvent, for example pyridine, tetrahydrofuran, dioxane, ethyl acetate, chloroform, methylene chloride, dimethylformamide or diethylacetamide, or in mixtures thereof, at room temperature or slightly elevated or reduced temperature, for example in a temperature range from about xe2x88x9210xc2x0 C. to about 50xc2x0 C., preferably at room temperature, the reaction in the presence of the dehydrating agent also being carried out at lower temperatures, for example at about 0xc2x0 C. 
In the preparation, according to the invention, of di-, tri- or tetra-nucleotides in step b), nucleosides of the formula V which are protected in the 5xe2x80x2-position and, if appropriate, in the base part and in which R1 and B are as defined above are reacted with activated phosphorus esters of the formula VII, in which X1 and X2 independently of one another are hydroxyl or salts derived therefrom, halogen, imidazolyl, 1,2,4-triazol-1-yl, tetrazolyl or 1-benzotriazolyloxy, and X2 additionally can also be 2-cyanoethoxy, 2-trihalogenoethoxy, 2-arylsulfonylethoxy, 2-lower alkylthioethoxy, 2-arylthioethoxy or 2-(4-nitrophenyl)-ethoxy and R2 is a protective group which can be detached by a base or nucleophiles, such as ammonium hydroxide, thiophenolate or an arylaldoximate, such as phenyl which is unsubstituted or substituted by halogen, nitro and/or lower alkyl, methyl or benzyl which is unsubstituted or substituted by nitro, or a protective group which can be detached by metal ions, such as 8-quinolyl or 5-chloro-8-quinolyl, in the presence or absence of dehydrating agents or in the presence or absence of bases.
A compound of the formula VIII formed in this manner, in which R1, X2 and R2 are as defined above, is subsequently first reacted, if appropriate, with a 2-substituted ethanol which converts the radical X2 into a group OR3, in which R3 is cyanoethyl, 2-trihalogenoethyl, 2-arylsulfonylethyl, 2-lower alkylthioethyl, 2-arylthioethyl or 2-(4-nitrophenyl)-ethyl, the protective group R1 is then detached and the compound of the formula IX prepared in this manner is reacted with another compound of the formula VIII in the presence or absence of dehydrating agents or in the presence or absence of bases, to give a dinucleotide X (equation 2). If appropriate, a compound of the formula VIII is converted into another compound of the formula VIII, in which X2 is hydroxyl or salts derived therefrom, by reaction with bases and water.
The reactions are carried out in one of the abovementioned inert solvents at room temperature or slightly elevated or reduced temperature, for example at room temperature.
The protective group R1 is detached, for example, with the aid of acids, such as mineral acids, for example hydrochloric acid or sulfuric acid, carboxylic acids, for example acetic acid, trichloroacetic acid or formic acid, sulfonic acids, for example methanesulfonic or p-toluenesulfonic acid, or, in particular, Lewis acids, for example zinc chloride, zinc bromide, aluminium chloride, dialkylaluminium halides, for example dibutyl- or diethyl-aluminium chloride, or boron trifluoride, at 10xc2x0 C. to 50xc2x0 C., in particular at room temperature. If a dialkylaluminium halide is used, the detachment is carried out in a lipophilic solvent, in particular in toluene, and if one of the other Lewis acids mentioned is used, in a solvent mixture, consisting of a halogenohydrocarbon, for example methylene chloride, and a lower alkanol, for example ethanol or isopropanol. 
The preparation, according to the invention, of dinucleotides of the formula X also comprises the reaction of nucleosides of the formula V, in which R1 and B are as defined above, with phosphites of the formula VIIA, in which X1 is halogen, in particular chlorine, X2 is halogen, in particular chlorine, di-lower alkylamino, in particular dimethylamino or diisopropylamino, or morpholino, piperidino or pyrrolidino, and R2 is as defined above for VII, and is, in particular, methyl, in the presence or absence of a suitable base. The compounds of the formula VIIIA obtainable according to the invention are reacted, on the one hand, with a 2-substituted ethanol, which converts the radical X2 into a group OR3, in which R3 is as defined above, and are then oxidized with an oxidizing agent, for example iodine, in the presence of a base to give the phosphate, and the protective group R1 is detached, a compound of the formula IX being formed, or, on the other hand, are reacted with a compound of the formula IX and are then oxidized with an oxidizing agent, for example iodine in the presence of a base, to give a compound of the formula X (equation 3). 
To prepare, according to the invention, trinucleotides, the protective group R1 is detached from dinucleotides of the formula X, in which R1, R2 and R3 are as defined above and in which B1 and B2 independently of one another are thymyl, costosyl, adenyl or guanyl, and the resulting compound is reacted with a compound of the formula VIII, in the presence or absence of dehydrating agents or in the presence or absence of bases, or with a compound of the formula VIIIA, with subsequent oxidation, a compound of the formula XI being formed (equation 4). The detachment of the protective group R1 and the condensation to give the trinucleotides of the formula XI are carried out in the same manner as that described for the preparation of the dinucleotides of the formula X. 
To prepare, according to the invention, tetranucleotides, trinucleotides of the formula XI are reacted as described above for dinucleotides of the formula X.
In a preferred embodiment of the invention, the 4-methoxytrityl group is used as the protective group R1, a phenyl group substituted by chlorine, in particular 2-chlorophenyl, is used as the protective group R2 and the 2-cyanoethyl group is used as the protective group R3. The 1-benzotriazolyloxy radical is the preferred radical X1 and X2 in the compound of the formula VII.
Trinucleotides of the formula XI are preferably prepared by detaching the protective group R1 from dinucleotides of the formula X and reacting the resulting compound with compounds of the formula VIII, in which X2 is hydroxyl or salts derived therefrom, in the presence of a dehydrating agent (equation 4). Examples of dehydrating agents according to the invention are 2,4,6-trimethyl- or -triisopropyl-benzenesulfonyl chloride, -imidazolide, -tetrazolide or -1,2,4-triazolide, unsubstituted or substituted by nitro. 2,4,6-Trimethylbenzenesulfonyl-3-nitro-1,2,4-triazolide of the formula XII 
is the preferred dehydrating agent.
Nucleosides in which the free amino group in the base part is protected are preferably used. Preferred protective groups are benzoyl for adenine, benzoyl or 4-methoxybenzoyl for cytosine, and isobutyryl or diphenylacetyl for guanine. Thymine is preferably used without a protective group.
An apparatus which is known per se and has a semi-automatic or fully automatic, microprocessor-controlled feed system for solvents and reagents is used in the preparation, according to the invention, of oligonucleotides in step c). The protective group R1 is detached, as described above, from a compound of the formula VI prepared according to step a), and the product is then reacted either with a compound of the formula VIII, or with a compound of the formula VIIIA, or with a compound of the formula X or XI, in which the protective group R3 has been detached beforehand with bases (a 2-cyanoethyl group R3 is detached, for example, with a tri-lower alkylamine, for example triethylamine, in one of the abovementioned inert solvents or solvent mixtures at 10xc2x0 C. to 40xc2x0 C., in particular at room temperature), in the presence or absence of a dehydrating agent or in the presence or absence of a base. The invention also relates to reactions in which a tetranucleotide prepared according to step b) is used instead of a dinucleotide of the formula X or a trinucleotide of the formula XI. If a phosphite of the formula VIIIA is used, after-treatment is subsequently carried out with an oxidising agent, for example iodine in the presence of a base. The compound of the formula XIII prepared in this manner, in which R1, R2 and B are as defined above and n is an integer from 1 to 4, is subjected to the reaction steps described for the compound of the formula VI (detachment of R11 reaction with VIII, VIIIA, X, XI or the corresponding tetranucleotide, if appropriate with oxidative after-treatment) as frequently as necessary until a compound of the formula XIII is formed, in which n is any selected number between about 19 and about 69. 
In a preferred embodiment of the invention, 4-methoxy-trityl is used as the protective group R1 and the detachment is carried out with zinc bromide in the presence of a CH- or NH-acid compound, in particular 1,2,4-triazole or tetrazole. The use of, for example, 1,2,4-triazole in the detachment of the 4-methoxytrityl protective group is novel and, surprisingly, leads to the detachment-proceeding rapidly, with high yields and without side reactions. It is particularly preferable to use zinc bromide and 1,2,4-triazole in a molar ratio of between 20:1 and 100:1 in a solvent mixture consisting of an aprotic solvent and an alcohol, for example methylene chloride and 2-propanol.
In a preferred embodiment of the invention, a compound of the formula VI or of the formula XIII, in which the lprotective group R1 has been detached, is reacted with a trinucleotide of the formula XI, in which the protective group R3 has been detached, in the presence of a dehydrating agent, for example 2,4,6-trimethyl- or -triisopropyl-benzene-sulfonyl chloride, -imidazolide, -tetrazolide or -1,2,4-triazolide, unsubstituted or substituted by nitro. 2,4,6-Trimethylbenzenesulfonyl-3-nitro-1,2,4-triazolide of the formula XII is particularly preferred.
The particularly preferred combination, which comprises using the 4-methoxytrityl group as the protective group R1, using zinc bromide in the presence of 1,2,4-triazole for the detachment of R1 and using the triazolide of the formula XII as the dehydrating agent for the reaction of the de-protected oligonucleotide/polystyrene resin of the formula XIII with a de-protected trinucleotide of the formula XI makes it possible, surprisingly, for long nucleotide chains with about 40 to about 70 bases also to be prepared in a short time, in high yields and in high purity.
Processes which are known per se are used for the detachment, according to the invention, of the oligodeoxynucleotides from the carrier and for the removal of the protective groups in step d). An arylaldoximate, for example 1,1,3,3-tetramethylguanidinium 2-nitrobenzaldoximate, is the particularly preferred reagent for detachment from the carrier and for removal of the preferred 2-chlorophenyl protective group. The reaction is carried out in one of the abovementioned inert solvents, to which a little water has been added, for example in 95% pyridine, at room temperature. The product is then reacted with aqueous ammonia at room temperature or elevated temperature, for example at 20xc2x0 C. to 70xc2x0 C., in particular at 50xc2x0 C.
For ligation of the oligodeoxynucleotides according to the invention, a phosphate radical is introduced at the 5xe2x80x2-terminal hydroxyl group. The introduction of the phosphate radical (phosphorylation) is carried out in a manner which is known per se, with the aid of T4 polynucleotide kinase in the presence of ATP.
Oligodeoxynucleotides, prepared according to the invention, from the coding and the complementary DNA strand contain overlapping sequences consisting of at least 3, preferably 8 to 15, overlapping base pairs. Such oligodeoxynucleotide pairs are held together by hydrogen bridge bonding during mixing. The overhanging, single-stranded ends serve, in step e1) and e2), as the matrix (template) for the build-up of the second (complementary) strand by a DNA-polymerase, for example DNA-polymerase I, the Klenow fragment of DNA-polymerase I or T4 DNA-polymerase, or with AMV reverse transcriptase, in the presence of the four deoxynucleoside triphosphates (dATp, dCTp, dGTp and TTP). The duplex-DNAs formed during complementing, which are, in particular, fragments of the (modified) eglin gene (process e1) or the complete (modified) eglin gene (process e2) have flat ends.
The fragments of the (modified) eglin gene which are obtainable by process step e1) contain, on their ends, nucleotide sequences which can be recognised and cleaved by restriction endonucleases. Depending on the choice of nucleotide sequences and accordingly the restriction endonucleases, completely base-paired (flat) ends (xe2x80x9cblunt endsxe2x80x9d) or ends with an overhanging DNA strand (xe2x80x9cstaggered endsxe2x80x9d) are formed during cleavage. The restriction recognition sequences are chosen so that the ligation of the DNA fragments which have been treated with a restriction endonuclease which forms blunt ends, or the base-pairing of the cohesive ends and the subsequent ligation of DNA fragments with staggered DNA strands produces the complete (modified) eglin structure gene. The ligation of two double-stranded DNA fragments requires a 5xe2x80x2-terminal phosphate group on the donor fragment and a free 3xe2x80x2-terminal hydroxyl group on the acceptor fragment. The DNA fragments obtained are already 5xe2x80x2-terminally phosphorylated and are linked with a ligase, in particular T4 DNA-ligase, in a manner which is known per se.
In a preferred embodiment of the present invention, two fragments of the eglin C or B gene, in the case of the eglin C gene in particular the fragments F1(C) and F2 according to formula IIIa or IV, and in the case of the eglin B gene in particular fragments F1(B) and F2 according to formula IIIb or IV, are prepared in the manner described. The fragments, which can be subcloned in a suitable vector if necessary, preferably contain in each case the recognition sequence for a restriction endonuclease, in particular HpaII, at the linking ends, which is why, after cleavage with the said restriction enzyme and ligation of the two fragments, the correctly coding eglin DNA sequence is formed. In addition, the fragment 1 before the translation start signal (ATG) and the fragment 2 after the translation stop signal (for example TAG) also contain xe2x80x9cterminalxe2x80x9d restriction sites which allow incorporation of the (modified) eglin gene or the (modified) eglin gene fragments into a suitable vector.
The invention particularly relates to the preparation of the eglin C gene in two fragments F1(C) and F2 of the formula IIIa and IV, which produce the correct eglin C DNA sequence after cleavage with the restriction enzyme HpaII and ligation, and in which F1(C) has an EcoRI restriction site before the translation start signal and F2 has a BamHI restriction site after the translation stop signal.
In another embodiment (step e2), in each case two oligodeoxynucleotides, which originate alternatively from the coding and the complementary strand, are fused by means of at least 3, preferably 8 to 15, complementary bases, made up with a DNA-polymerase, for example one of those mentioned above, and ligated with T4 DNA-ligase to give the (modified) eglin structure gene.
Preparation of Expression Vectors Containing an Eglin Gene
The invention furthermore relates to expression vectors which contain a DNA sequence which codes an eglin or a modified eglin and which is regulated by an expression control sequence such that polypeptides with eglin activity are expressed in a host transformed with these expression vectors.
The expression vectors according to the present invention contain a sequence which codes eglin B, modified eglin B, modified eglin C or, in particular, eglin C.
The expression vectors of the present invention are prepared, for example, by inserting a DNA sequence which codes an eglin or a modified eglin into a vector-DNA, which contains an expression control sequence, such that the expression control sequence regulates the said DNA sequence.
A suitable vector is chosen from the host cells envisaged for transformation. Examples of suitable hosts are microorganisms, such as yeasts, for example Saccharomyces cerevisiae, and, in particular, strains of bacteria which do not have restriction enzymes or modification enzymes, in particular strains of Escherichia coli, for example E. coli X1776, E. coli HB101, E. coli W3110, E. coli HB101/LM1035, E. coli JA221(37) or E. coli K12 strain 294, Bacillus subtilis, Bacillus stearothermophilus, Pseudomonas, Haemophilus, Streptococcus and others, and furthermore cells of higher organisms, in particular established human or animal cell lines. The above strains of E. coli, for example E. coli HB101 and E. coli JA221, and furthermore Saccharomyces cerevisiae are preferred as the host microorganism.
In principle, all vectors which replicate and express the DNA sequences according to the invention in the chosen host are suitable.
Examples of vectors which are suitable for the expression of an eglin or modified eglin gene in an E. coli strain are bacteriophages, for example derivatives of xcex bacteriophages, or plasmids, such as, in particular, the plasmid co1E1 and its derivatives, for example pM89, pSF2124, pBR317 or pBR322. The preferred vectors of the present invention are derived from plasmid pBR322. Suitable vectors contain a complete repticon and a labelling gene, which makes it possible to select and identify the hosts transformed with the expression plasmids on the basis of a phenotypical characteristic. Suitable labelling genes impart to the host, for example, resistance towards heavy metals, antibiotics and the like. Furthermore, preferred vectors of the present invention contain, outside the replicon and labelling gene regions, recognition sequences for restriction endonucleases, so that the eglin gene and, if appropriate, the expression control sequence can be inserted at these sites. The preferred vector, the plasmid pBR322, contains an intact replicon, labelling genes which impart resistance towards tetracycline and ampicillin (tetR and ampR) and a number of recognition sequences, occurring only once, for restriction endonucleases, for example PstI (cleaves in the ampR gene, the tetR gene remains intact), BamHI, HindIII and SaLI (all cleave in the tetR gene, the ampR gene remains intact), NruI and EcoRI.
Several expression control sequences can be used for regulation of the gene expression. In particular, expression control sequences of highly expressed genes of the host to be transformed are used. In the case of pBR322 as the hybrid vector and E. coli as the host microorganism, for example, the expression control sequences (which contain, inter alia, the promoter and the ribosomal bonding site) of the lactose operon, tryptophan operon, arabinose operon and the like, the xcex2-lactamase gene, the corresponding sequences of the phage xcexN gene or the phage fd-stratified protein gene and others, are suitable. Whilst the plasmid peR322 already contains the promoter of the xcex2-lactamase gene (xcex2-lac-gene), the other expression control sequences must be introduced into the plasmid. The preferred expression control sequence in the present invention is that of the tryptophan operon (trp po).
Vectors which are suitable for replication and expression in yeast contain a yeast replication start and a selective genetic marker for yeast. Hybrid vectors which contain a yeast replication start, for example chromosomal autonomously replicating segment (ars), are retained extrachromosomally within the yeast cell after the transformation and are replicated autonomously during mitosis. Furthermore, hybrid vectors which contain sequences homologous to the yeast-2xcexc-plasmid-DNA can be used. Such hybrid vectors are incorporated by recombination within the cell of already existing 2xcexc-plasmids, or replicate autonomously. 2-sequences are particularly suitable for plasmids with a high transformation frequency and permit a high number of copies. Suitable labelling genes for yeasts are, in particular, those which impart antibiotic resistance to the host or, in the case of auxotrophic yeast mutants, genes which complement host defects. Corresponding genes impart, for example, resistance towards the antibiotic cycloheximide or ensure prototrophy in an auxotrophic yeast mutant, for example the URA3, LEU2, HIS3 or, in particular, TRP1 gene. Yeast hybrid vectors furthermore preferably contain a replication start and a labelling gene for a bacterial host, in particular E. coli, so that the construction and cloning of the hybrid vectors and their intermediates can take place in a bacterial host. Expression control sequences which are suitable for expression in yeast are, for example, those of the TRP1, ADHI, ADHII, PH03 or PH05 gene, and furthermore promoters involved in glycolytic degradation, for example the PGK and the GAPDH promoter.
The invention particularly relates to expression vectors which are capable of replication and phenotypical selection and which contain an expression control sequence and a DNA sequence which codes an eglin or a modified eglin, the said DNA sequence together with the transcription start signal and termination signal and the translation start signal and stop signal being arranged in the said expression plasmid under regulation of the said expression control sequence such that polypeptides with eglin activity are expressed in a host transformed with the said expression plasmid.
In order to achieve effective expression, the structure gene must be arranged correctly (in xe2x80x9cphasexe2x80x9d) with the expression control sequence. It is advantageous for the expression control sequence to be linked with the eglin (or modified eglin) gene, which preferably contributes its own translation start signal (ATG) and translation stop signal (for example TAG), in the region between the main mRNA start and the ATG of the gene-coding sequence, which is of course linked with the expression control sequence (for example the xcex2-lac-coding sequence when thexcex2-lac promoter is used). Effective transcription and translation are thereby ensured.
For example, a vector, in particular pBR322, is cleaved with a restriction endonuctease and, if appropriate after modification of the linearised vector thus formed, an expression control sequence provided with corresponding restriction ends is introduced. The expression control sequence contains the recognition sequence of a restriction endonuclease at the 3xe2x80x2-end (in the translation direction), so that the vector already containing the expression control sequence can be digested with the said restriction enzyme and the eglin (or modified eglin) structure gene provided with appropriate ends can be inserted. A mixture of two hybrid plasmids containing the gene in correct and incorrect orientation is thereby formed. It is advantageous also to cleave the vector already containing the expression control sequence with a second restriction endonuclease within the vector-DNA and to insert the structure gene provided with correct ends in the resulting vector fragment. All the operations on the vector are preferably carried out such that the function of the replicon and at least one labelling gene is not impaired.
In a preferred embodiment of the present invention, a vector derived from pBR322, which contains an expression control sequence, in particular that of tryptophan operon (trp po), which carries at the 3xe2x80x2-end (between the main mRNA start and the first ATG), the recognition sequence for a restriction endonuclease, which preferably forms cohesive ends, for example EcoRI, is digested with the restriction endonuclease mentioned and, in the vector-DNA part, with a second restriction endonuclease which forms blunt or, preferably, cohesive ends, for example BamHI, after which the vector thus linearised links with the eglin (or modified eglin) gene containing the appropriate ends (for example with an EcoRI end before the ATG start and a BamHI end after the translation stop codon). Linking is effected in the known manner, by pairing of the complementary (cohesive) ends and ligation, for example with T4-DNA-ligase.
The eglin (or modified eglin) gene obtained via the mRNA route, from genomic DNA or synthetically and provided with corresponding cohesive (in particular EcoRI and BamHI) ends can also be cloned in a vector, for example pBR322, before introduction into an expression plasmid, in order to obtain larger amounts of structure gene, for example for sequence analysis. The cLones containing the hybrid plasmid are isolated, for example, with an eglin-specific, radio-actively labelled oligodeoxynucleotide probe (see above). The eglin (or modified eglin) gene is characterised, for example, by the method of Maxam and Gilbert (3).
In a preferred embodiment of the invention, two fragments of an eglin or modified eglin gene, for example two fragments of the eglin C gene, are synthesised. Fragment 1, which includes the 1st part of the gene, contains, before the ATG and at the end, in each case the recognition sequence for restriction endonucleases which form cohesive ends, for example EcoRI before the ATG and HpaII at the end. Fragment 2, which includes the rear part of the gene, has corresponding recognition sequences, for example HpaII at the start, and BamHI after the translation stop signal (for example TAG). The fragments are cleaved at the outer recognition sequences (fragment 1, for example, with EcoRI and fragment 2 correspondingly with BamHI) and are subcloned in a correspondingly cleaved vector (for example pBR322). The identification of the clones containing the fragments and the characterisation of the fragments are carried out as described above. The fragments are then excised from the hybrid vectors with the corresponding restriction endonucleases (fragment 1, for example, with EcoRI and HpaII and fragment 2, for example, with HpaII and BamHI) and are Ligated via their cohesive ends, in particular their HpaU ends, whereupon the complete eglin (or modified eglin) gene is formed, this gene being inserted, as described, into a vector-DNA.
Transformation of the Host Cells
The invention also reLates to a process for the preparation of a transformed host, which comprises transforming a host with an expression plasmid containing a DNA sequence which is regulated by an expression control sequence and codes an eglin or a modified eglin.
Examples of suitable hosts are the abovementioned microorganisms, such as strains of Saccharomyces cerevisiae, Bacillus subtilis and, in particular, Escherichia coli. The transformation with the expression plasmids according to the invention is carried out, for example, as described in the literature, thus for S. cerevisiae (4), B. subtilis (5) and E. coli (6). The transformed host is advantageously isolated from a selective nutrient medium, to which the biocide against which the labelling gene contained in the expression plasmid imparts resistance is added. If, as preferred, the expression plasmids contain the ampR gene, ampicillin is accordingly added to the nutrient medium. Cells which do not contain the expression plasmid are destroyed in such a medium.
The invention also relates to the transformed host obtainable by the route described.
Culture of the Transformed Host and Production of Eglins
The transformed host can be used for the preparation of eglins and modified eglins. The process for the preparation of eglins and modified eglins comprises culturing the transformed host and releasing the product from the host cells and isolating it.
Surprisingly, it has now been found that the transformed hosts according to the invention produce mixtures of polypeptides with eglin activity. Natural eglins, methionyl-eglins and N-terminally acetylated or shortened eglins can be isolated from the mixtures in varying ratios, depending on the host microorganism used and the cultivation conditions applied. Thus, one important product which can be isolated from transformed E. coli strains and from transformed yeast differ from the natural eglins B and C by an N-acetyl radical on the N-terminal aminoacid threonine. The production of Nxcex1-acetylated products is particularly surprising. In particular, the production of such polypeptides by means of genetic engineering methods has not yet hitherto been observed. Thus, even xcex1-thymosin, which is naturally N-terminally acetylated, is expressed in the non-acetylated form by corresponding genetically modified hosts (35).
The production of N-terminally acetylated eglins is of great advantage, because such compounds have an increased stability towards the aminopeptidases present in the host cells, which means that (partial) proteolytic degradation starting from the N-terminus is prevented and as a result the yield is increased. Furthermore, the purification process is thereby considerably simplified, because the desired products are not contaminated with fragments formed by proteolytic degradation.
The present invention thus furthermore relates to a process for the preparation of eglin compounds of the formula
(Met)r-B-ProGluValValGlyLysThrValAspGlnAlaArgGluTyrPheThrLeuHisTyrProGlnTyrAspValWPheLeuProGluGlySerProValThrLeuAspLeuArgTyrAsnArg ValArgValPheTyrAsnProGlyThrAsnValValAsn-Bxe2x80x2xe2x80x83xe2x80x83(XIV)
in which B is a direct bond or a peptide radical comprising 1-10 aminoacid units from the N-terminus of the natural eglins, for example such a radical chosen from the group comprising SerPhe, LeuLysSerPhe, SerGluLeuLysSerPhe, PheGlySerGluLeuLysSerPhe and ThrGluPheGlySerGLuLeuLysSerPhe, and Bxe2x80x2 is not a peptide radical or is a peptide radical which comprises 1-6 aminoacid units from the C-terminus of the naturat eglins, for example such a radical chosen from the group comprising HisVal, HisValProHis or HisValProHisValGly, W is Tyr or His and r is 0 or 1, and in which, in compounds of the formula XIV in which r is 0, the N-terminal aminoacid is free or N-acetylated, and of salts of such compounds, which comprises culturing a host transformed with an expression plasmid containing an eglin-coding DNA sequence regulated by an expression control sequence, in a liquid nutrient medium containing assimilatable sources of carbon and nitrogen, releasing the product from the host cells and isolating it, or, for the preparation of compounds of the formula XIV, in which r is 0 and the N-terminal aminoacid is N-acetylated, acetylating a compound of the formula XIV with a free N-terminal amino group and, if desired, converting an eglin compound of the formula XIV, which can be obtained, into another eglin compound of the formula XIV, and, if necessary, separating a mixture, obtainable according to the process, of compounds of the formula XIV into the individual components, and/or, if desired, converting a resulting salt into the free polypeptide and converting a resulting polypeptide into a salt thereof.
The invention preferably relates to a process for the preparation of eglin compounds of the formula
VGluPheGlySerGluLeuLysSerPheProGluValValGlyLysThrValAspGlnAlaArgGluTyrPheThrLeuRisTyrProGlnTyrAspValWPheLeuProGluGlySerProValThrLeuAspLeuArgTyrAsnArgValArgValPheTyrAsnProGlyThrAsnValValAsnHisValProHisValGly xe2x80x83xe2x80x83(XIVxe2x80x2)
in which V is Thr, N-acetyl-Thr or Met-Thr and W is Tyr or His, and of salts of such compounds, which comprises culturing a host microorganism transformed with an expression plasmid containing an eglin-coding DNA sequence regulated by an expression control sequence, in a liquid nutrient medium containing assimitatable sources of carbon and nitrogen, releasing the eglin from the microorganism cells and isolating it, and, if desired, converting an eglin which can be obtained, in which V is N-acetyl-Thr or Met-Thr and W has the above meaning, into an eglin in which V is Thr, and, if necessary, separating a mixture, obtainable according to the process, of compounds of the formula XIV into the individual components, and/or, if desired, converting a resulting salt into the free polypeptide or a resulting polypeptide into a salt thereof.
The invention particularly relates to a process for the preparation of eglin C compounds of the formula XIV, in which B is a peptide radical selected from the group comprising LeuLysSerphe, SerGluLeuLysSerPhe, PheGlySerGluLeuLysSerPhe and ThrGLuPheGlySerGluLeuLysSerPhe, Bxe2x80x2 is the radical -HisValProHisVatGly, W is Tyr and r is 0 or 1, and furthermore also a process for the preparation of eglin B compounds of the formula XIV, in which B is the peptide radical ThrGluPheGlySerGLuLeuLysSerPhe, Bxe2x80x2 is the peptide radical -HisValProHisValGly, W is His and r is 0 or 1, the N-terminal aminoacid in compounds of the formula XIV in which r is 0 being free or N-acetylated, and of salts of such compounds.
In the compounds of the formula XIV, W is preferably Tyr (eglin C compounds).
The invention particularly relates to a process for the preparation of eglin C compounds of the formula XIV, in which B is the PheGlySerGluLeuLysSerPhe, ThrGLuPheGlySerGluLeuLysSerPhe or N-acetyl-ThrGluPheGlySerGluLeuLysSerphe radical, Bxe2x80x2 is the-HisValProHisValGly radical, W is Tyr and r is 0, and of salts of such compounds.
The invention especially relates to a process for the preparation of eglin C, N-methionyl-eglin C, N-acetyl-eglin C, the modified eglin C compound Des-eglin C, and the modified eglin C compound eglin Cxe2x80x2 and eglin Cxe2x80x3.
Various sources of carbon can be used for culture of the transformed hosts according to the invention. Examples of preferred sources of carbon are assimilatable carbohydrates, such as glucose, maltose, mannitol or lactose, or an acetate, which can be used either by itself or in suitable mixtures. Examples of suitable sources of nitrogen are aminoacids, such as casaminoacids, peptides and proteins and their degradation products, such as tryptone, peptone or meat extracts; and furthermore yeast extracts, malt extract and also ammonium salts, for example ammonium chloride, sulfate or nitrate, which can be used either by themselves or in suitable mixtures. Inorganic salts which can also be used are, for example, sulfates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium.
The medium furthermore contains, for example, growth-promoting substances, such as trace elements, for example iron, zinc, manganese and the like, and preferably substances which exert a selection pressure and prevent the growth of ceLls which have lost the expression plasmid. Thus, for example, ampicillin is added to the medium if the expression plasmid contains an ampR gene. Such an addition of antibiotic substances also has the effect that contaminating antibiotic-sensitive microorganisms are destroyed.
Culture is effected by processes which are known per se. The culture conditions, such as temperature, pH value of the medium and fermentation time, are chosen so that a maximum eglin titre is obtained. Thus, an E. coli strain is preferably cultured under aerobic conditions by submerse culture with shaking or stirring at a temperature of about 20 to 40xc2x0 C., preferably about 30xc2x0 C., and a pH value of 4 to 9, preferably at pH 7, for about 4 to 20 hours, preferably 8 to 12 hours. The expression product (eglin) thereby accumulates intracellularly.
When the cell density has reached a sufficient value, the culture is interrupted and the eglin is released from the cells of the host. For this purpose, the cells are destroyed, for example by treatment with a detergent, such as SDS or triton, or Lysed with lysozyme or a similarly acting enzyme. Alternatively or additionally, mechanical forces, such as shearing forces (for example X-press, French press, Dyno mill) or shaking with glass beads or aluminium oxide, or alternating freezing, for example in liquid nitrogen, and thawing, for example to 30xc2x0 to 40xc2x0 C., as well as ultra-sound can be used to break the cells. The resulting mixture, which contains proteins, nucleic acids and other cell constituents, is enriched in proteins, including eglin, in a manner which is known per se, after centrifugation. Thus, for example, most of the non-protein constituents are removed by polyethyleneimine treatment and the proteins, including eglin, are precipitated, for example, by saturation of the solution with ammonium sulfate or with other salts. Bacterial proteins can also be precipitated by acidification with acetic acid (for example 0.1%, pH 4-5). Further enrichment of eglin can be achieved by extraction of the acetic acid supernatant liquor with n-butanol. Further purification steps include, for example, gel electrophoresis, chromatographic processes, such as ion exchange chromatography, size exclusion chromatography, HPLC, reverse phase HPLC and the like, separation of the constituents of the mixture according to molecular size by means of a suitable Sephadex column, dialysis, affinity chromatography, for example antibody, especially monoclonal antibody, affinity chromatography or affinity chromatography on an anhydrochymotrypsin column, and other known processes, especially those known from the literature.
Isolation of the expressed eglins comprises, for example, the following stages: removal of the cells from the culture solution by means of centrifugation; preparation of a crude extract by destruction of the cells, for example by treatment with a lysing enzyme and/or alternating freezing and rethawing; removal of the insoluble constituents by centrifugation; precipitation of the DNA by addition of polyethyleneimine; precipitation of the proteins, including eglin, by ammonium sulfate; affinity chromatography of the dissolved precipitate on a monoclonal anti-eglin antibody column or an anhydrochymotrypsin column; demineralisation of the resulting solution by means of dialysis or chromatography on Sephadex G25.
Alternatively, after the DNA has been separated off, the bacterial proteins can be precipitated with 0.1% acetic acid and the eglin can be extracted from the acid supernatant liquor with n-butanol or the acid supernatant liquor can be subjected directly to ion exchange chromatography (for example on carboxymethylcellulose). Further purification steps include gel filtration on Sephadex G50 (or G75) and reverse phase HPLC. Demineralisation is again carried out on Sephadex G25.
The test with anti-eglin antibodies (for example monoclonal antibodies obtainable from rabbits or from hybridoma cells) or the inhibition of the proteases human leucocyte elastase (HLE) or cathepsin G (cat G) (1) by eglin can be used to detect the eglin activity.
The conversion of a compound of the formula XIV, in which r is 0 and the N-terminal amino group is in the free form, into a corresponding compound of the formula XIV, in which the N-terminal aminoacid is N-acetylated, is effected, in particular, by an enzymatic route. Thus, the introduction of the acetyl group can be carried out, for example, with the aid of an Nxcex1-acetyl-transferase (in the pure form, as an extract or lysate of a suitable microorganism or as an organ extract), for example from E. coli, from rabbit reticulocytes or wheat seedlings (8), in the presence of acetyl-coenzyme A.
Compounds of the formula XIV obtainable according to the process can be converted into other compounds of the formula XIV in a manner which is known per se.
Thus, methionine or the acetyl radical can be detached from compounds of the formula XIV, which can be obtained, with methionine as the N-terminal aminoacid or with an N-terminably acetylated amino group. For example, eglin compounds obtainable according to the invention with an N-terminal methionyl radical can be converted into eglins without such a radical by detaching the terminal methionyl radical by means of cyanogen bromide in the usual manner. The reaction with cyanogen bromide is carried out, for example, in an aqueous-acid medium, for example in very dilute hydrochloric acid, for example in 0.1-0.3 N hydrochloric acid, or in a strong organic acid, for example in 50-70% formic acid, at room temperature or slightly elevated or reduced temperature, for example at about 15xc2x0 to about 25xc2x0 C., over a period of about 24 hours. The acetyl radical can correspondingly be detached from compounds of the formula XIV, obtainable according to the process, with an N-terminally acetylated amino group. The detachment of the acetyl radical can be carried out, for example, enzymatically, such as with suitable acylases, for example from pigs"" kidneys or from suitable microorganisms, or with suitable acetyl-transferases in the presence of coenzyme A, it also being possible to use extracts or lysates from microorganisms or organ extracts containing such enzymes instead of pure enzyme products (for example an E. coli HB101 lysate when E. coli HB101 is used as the strain producing Nxcex1-acetyl-eglin B or C).
A mixture, obtainable according to the process, of compounds of the formula XIV, for example consisting of compounds of the formula XIV, in which V is either Thr or acetyl-Thr, can be separated into the individual components in a manner which is known per se.
Examples of suitable separation methods are chromatographic processes, for example adsorption chromatography, ion exchange chromatography, HPLC or reversed phase HPLC, and furthermore multiplicative distribution or electrophoretic methods, for example electrophoresis on cellulose acetate or gel electrophoresis, in particular polyacrylamide gel electrophoresis (xe2x80x9cPAGExe2x80x9d).
The invention also relates to the novel peptides with eglin activity, which are obtainable by the process according to the invention, mixtures of such peptides and salts of such compounds.
The invention furthermore relates to the novel compounds of the formula
(Met)r-B-ProGluValValGlyLysThrValAspGlnAlaArgGlu TyrPheThrLeuHisTyrProGlnTyrAspValWPheLeuProGluGlySerProValThrLeuAsp LeuArgTyrAsnArgValArgValPheTyrAsnProGlyThrAsnValValAsn-Bxe2x80x2
in which r is 1, B is a direct bond or a peptide radical comprising 1-10 aminoacid units from the N-terminus of the natural eglins, for example a radical selected from the group comprising SerPhe, LeuLysSerPhe, SerGluLeuLysSerPhe, PheGlySerGluLeuLysSerPhe and ThrGluPheGlySerGluLeuLysSerPhe, and Bxe2x80x2 is not a peptide radical or is a peptide radical comprising 1-6 aminoacid units from the C-terminus of the natural eglins, for example such a radical selected from the group comprising -HisVal, -HisValProHis and -HisVaLProHisValGly, and W is Tyr or His, or in which r is 0, B is PheGlySerGLuLeuLysSerPhe or an N-terminally acetylated peptide radical, for example selected from the group comprising N-acetyl-SerPhe, N-acetyl-LeuLysSerPhe, N-acetyl-SerGluLeuLysSerPhe, N-acetyl-PheGLySerGluLeuLysSerPhe and N-acetyl-ThrGLuPheGLySerGLuLeuLysSerPhe, Bxe2x80x2 is as defined and W is Tyr or His, and salts of such compounds.
The invention particularly relates to compounds of the formula XIV, in which r is 0, B is the peptide radical PheGlySerGLuLeuLysSerPhe or N-acetyl-ThrGluPheGLySerGluLeuLysSerPhe, Bxe2x80x2 is the peptide radical-HisValProHisVaLGly and W is Tyr, and salts of such compounds.
The invention preferably relates to eglin compounds of the formula
VGluPheGlySerGluLeuLysSerPheProGluValValGlyLysThrValAspGlnAlaArgGlu TyrPheThrLeuHisTyrProGlnTyrAspValWPheLeuProGluGlySerProValThrLeuAsp LeuArgTyrAsnArgValArgValPheTyrAsnProGlyThrAsnValValAsnHisvalProHis ValGly (XIVxe2x80x2),
in which V is N-acetyl-Thr or Met-Thr and W is Tyr or His, and salts of such compounds.
The invention particularly relates to Nxcex1-acetyl-eglin C and salts thereof.
The compounds which can be prepared according to the invention and the novel compounds of the formula XIV can be not only in the free form, but also in the form of their salts, in particular their pharmaceutically acceptable salts. Since they contain several aminoacid radicals with free amino groups or guanidino groups, the compounds according to the invention can be, for example, in the form of acid addition salts. Possible acid addition salts are, in particular, physiologically acceptable salts with the usual therapeutically useful acids; inorganic acids are the hydrogen halide acids (such as hydrochloric acid), and also sulfuric acid and phosphoric or pyrophosphoric acid; suitable organic acids are, in particular, sulfonic acids (such as benzene- or p-toluene-sulfonic acid or lower alkanesulfonic acids, such as methane-sulfonic acid) and carboxylic acids, such as acetic acid, lactic acid, palmitic and stearic acid, malic acid, tartaric acid, ascorbic acid and citric acid. Since the eglin compounds also contain aminoacid radicals with free carboxyl groups which impart acid character to the entire peptide, they can also be in the form of a metal salt, in particular an alkali metal or alkaline earth metal salt, for example a sodium, potassium, calcium or magnesium salt, or an ammonium salt, derived from ammonia or a physiologically acceptable organic nitrogen-containing base. However, since they contain free carboxyl groups and free amino (and amidino) groups at the same time, they can also be in the form of an inner salt.
Depending on the procedure, the compounds according to the invention are obtained in the free form or in the form of acid addition salts, inner salts or salts with bases. The free compounds can be obtained from the acid addition salts in a manner which is known per se. Therapeutically acceptable acid addition salts or metal salts can in turn be obtained from the tatter by reaction with acids or bases, for example with those which form the abovementioned salts, and evaporation or lyophitisation. The inner salts can be obtained by adjusting the pH to a suitable neutral point.
Monoclonal Antibodies Against Eglins and Test Kits Containing Such Antibodies
The property of antibodies of binding specific antigens finds practical application outside the body in the quantitative determination (immunoassay) and in the purification of antigens (immunoaffinity chromatography). Serum from immunised animals usually contains a large number of various antibodies which react with the same antigen at various binding sites with various affinities, but in addition also antibodies against other antigens which reflect the earlier experiences of the individual. The successful use of antibodies for the determination and purification of antigens, however, requires high specificity and reproducibility.
Homogeneous antibodies which fulfill these requirements have been made accessible by the hybridoma technique described by Kxc3x6hler and Milstein (26). In principle, the technique comprises fusing antibody-secreting B lymphocytes, for example from the spleen, of immunised animals with tumour cells. The hybridoma cells formed combine the ability to multiply by division without limitation with the ability to form and secrete a homogeneous type of antibody. By culture in a selective medium in which non-fused tumour cells die but hybridoma cells multiply, and by suitable manipulation, it is possible to obtain and culture clones, i.e. cell populations, which are derived from a single hybridoma cell and are genetically identical, and to isolate the monoclonal antibodies produced by the cells.
The present invention relates to monoclonal antibodies against eglins or modified eglins, hybridoma cells which produce such antibodies, and processes for their preparation. Hybridoma cell lines and the monoclonal antibodies secreted from these which react specifically with eglin B or eglin C or derivatives thereof, for example Nxcex1-acetyl-eglin C or B or Nxcex1-methionyl-eglin C or B, are preferred. The process for the preparation of monoctonal anti-eglin antibodies comprises immunising mice with an eglin or modified eglin, fusing B lymphocytes from animals immunised in this manner with myeloma cells, cloning the hybridoma cells formed, then culturing the clones in vitro or by injection into mice and isolating antibodies from the cultures.
The invention furthermore relates to immunoaffinity chromatography columns and test kits for immunoassays containing these antibodies.
In the process according to the invention, mice, for example Balb/c mice, are immunised in a manner which is known per se but which is specific. Surprisingly, the immunisation is successful, even though eglins are relatively small protein molecules. In a preferred embodiment, a solution of 50 to 500 xcexcg, preferably 100 xcexcg, of eglin B or C, in particular in complete and incomplete Freund""s adjuvant and in buffered salt solution, is injected subcutaneously approximately every week or also at longer intervals over several weeks, for example 5 to 12 weeks, until a sufficient number of antibody-producing B lymphocytes has formed.
Organs containing B lymphocytes, for example spleen cells, are removed from the immunised mice and fused with those myeloma cells which, because of mutation, do not grow in a selective culture medium. Such myeloma cells are known and are, for example, those with the designation X63-Ag8, X63-Ag8.6.5.3, MPC-11, NS1-Ag4/1, MOPC-21 NS/1 or, in particular, SP 2/0. In a preferred embodiment, spleen cells from immunised mice are fused with myeloma cells of the cell line SP 2/0.
The fusion is carried out by processes known per se, by mixing the B lymphocytes and the myeloma cells, with the addition of a cell fusion agent, such as polyethylene glycol, Sendai virus, calcium chloride or lysolecithin. Fusion is preferably effected in the presence of polyethylene glycol, for example with a molecular weight of 500. After the fusion, the hybrids formed are cultured by a process which is known per se, in a selective culture medium complemented by hypoxanthine, aminopterin and thymidine (HAT medium). Non-fused myeloma cells cannot grow in this medium and die, as do normal lymphocytes.
The supernatant liquors from the hybridoma cultures can be tested for their content of specific antibodies by processes which are known per se, for example by radioimmunoassay or by agglutination. It is found here, surprisingly, that hybridoma cells which secrete antibodies specificalLy against eglin B or eglin C can be obtained by the process described. These antibodies also react with Nxcex1-acetyl-eglin C and B and Nxcex1-methionyl-eglin C and B.
The hybridoma cells which produce antibodies of the desired specificity are selected out, by cloning, from the mixture of the most diverse hybridoma cells resulting from the fusion. For this, cultures are started from a single growing cell by a process which is known per se, called xe2x80x9climiting dilutionxe2x80x9d.
The three hybridoma cell lines deposited at the Pasteur Institute, Paris, France under the designation 299S18-20, 299S22-1 and 299S22-10 can be obtained in this manner.
For mass production, the hybridoma cell clones which produce antibodies of the desired specificity are either cultured in vitro in media which are known per se or are injected into mice, for multiplication. In a preferred embodiment, hybridoma cells are injected into mice pretreated with pristane, ascites fluid is withdrawn and antibodies are isolated therefrom by precipitation with ammonium sulfate solution.
The monoclonal antibodies obtained with the aid of these hybridoma cells can be used in a manner which is known per se for the preparation of immunoaffinity chromatography columns. In a preferred embodiment of the invention, an antibody solution is added to a suitable carrier material (suspended in a buffer solution), non-bound constituents are then washed out and unoccupied sites of the carrier material are blocked.
The monoclonal antibodies obtained with the aid of the hybridoma cells can be used in a manner which is known per se for the preparation of test kits. These test kits can be based on various methods, for example on radioimmuno-diffusion, latex agglutination, spot tests, competitive or sandwich radioimmunoassay, enzyme immunoassay, immunofluorescence or immunochemical enzyme tests, for example ELISA or tandem ELISA. Besides the usual antibodies of various origins, such kits can contain antibody conjugates with enzymes or fluorescence carriers, and in addition an eglin or modified eglin, for example eglin B, eglin C or Nxcex1-acetyl-eglin C, Labelled with radioactive isotopes, such as I125, or conjugated with enzymes, for example with horseradish peroxidase or alkaline phosphatase, and furthermore enzyme substrates, suitable buffers, gels, latex, polystyrene or other filling materials and carriers.
The serological tests can be carried out, for example, as follows: Besides competitive RIA, a direct bonding test can be utilised to establish anti-eglin C antibody activity. For this purpose, eglin C is fixed in depressions in microtitre plates (200 ng/depression) by incubation overnight and then incubated with hybridoma culture fluid and rendered visible with goat anti-mouse Ig antibodies either radioactively labelled with 125I (solid phase RIA) or labelled by alkaline phosphatase (solid phase ELISA).
The three monoclonal antibodies selected are suitable for non-radioactive tandem ELISA, with the aid of which eglins can be determined quantitatively in body fluids.
The suitable pairs of antibodies were selected as follows, by means of competitive RIA: The monoclonal antibodies 299S18-20, 299S22-1 and 299S22-10 (200-300 ng/depression, obtained by ammonium sulfate precipitation from ascites fluid) and a polyclonal rabbit anti-eglin C antibody (200-300 ng/depression, obtained from serum) are fixed in depressions of microtitre plates. Inhibition of the bonding of 125I-labelled eglin C was investigated crosswise.
The experiments showed that the monoclonal antibodies 299S18-20 and 299S22-10 inhibit one another in bonding to eglin C, from which it can be concluded that they both bond to the same epitopes on the eglin C molecule.
The monoclonal antibodies 299S18-20 and 299S22-1 do not inhibit one another. This means that they bond to different epitopes of the eglin C molecule.
The relative bonding capacity, determined by the amount of fixed radioactively labelled eglin C bonded by the fixed antibodies, is highest with 299S22-10 and lowest with 299S18-20.
On the basis of tandem ELISA experiments, in which the monoclonal antibodies were tested in pairs, one antibody always being fixed as the solid phase on microtitre plates and the other being labelled, as the liquid phase, with an enzyme, for example alkaline phosphatase, it was found that the pairs 299S18-20/299S22-1 and 299S22-1/299S22-10, which are not cross-reactive, are most suitable for such quantitative assay, it being necessary for in each case the first of the monoclonal antibody pairs mentioned, which bonds weakly to eglin C, to be used as the solid phase.
The monoclonal antibodies according to the invention, as the solid phase, can also be used for the quantitative determination of eglin C together with a polyclonal anti-eglin C antibody, for example from sheep, as the liquid phase.
The sensitivity of the tandem ELISA is about 1-10 ng of eglin C/ml of a sample.
Pharmaceutical Products
The known (for example eglin B and eglin C) and novel (for exampLe Nxcex1-acetyl-eglin B and -eglin C and methionyl-eglin C and -eglin B) eglins and modified eglins obtainable according to the present invention have useful pharmacological properties and, like the eglins extracted from leeches (cf. German Offenlegungsschrift 2,808,396), can be used prophylacticalty or, in particular, therapeuticalLy.
The novel eglin compounds according to the invention, such as Nxcex1-acetyl-eglin B and Nxcex1-acetyl-eglin C, are distinguished by a very potent and specific inhibition of human Leucocyte elastase (HLE), teucocyte cathepsin G (H.cat.G) and chymotrypsin. The association rate constants (kass) and the equilibrium constants (Ki) of the enzyme-inhibitor complexes formed for the reactions of Nxcex1-acetyl-eglin C and two naturally occurring protease inhibitors, xcex11-proteinase inhibitor (xcex11PI, previously called xcex11-antitrypsin) and xcex12-macroglobulin (xcex12M), with HLE and H.cat.G are summarised in the following table:
Conditions: The association rate constants were determined by the method of Bieth et at. (36). The ki values for the interaction of Nxcex1-acetyl-eglin C with HLE and H.cat.G were catculated from xe2x80x9csteady statexe2x80x9d reaction rates, on the assumption that these interactions are reversible. All the values were determined at 37xc2x0 C. and pH 7.4.
The data show that the association rate constants for the reaction of Nxcex1-acetyl-eglin C and the natural inhibitors xcex11PI and xcex12M with HLE or H.cat.G are of the same order of magnitude. The high stability of the Nxcex1-acetyl-eglin/enzyme complexes (ki values!), the proven extremely tow toxicity of the eglins and their specificity (no significant interactions are observed with other mammalian proteases, in particular with those of the blood coagulation, fibrinolysis and complement systems), their increased stability towards proteolytic degradation by aminopeptidases due to the N-terminal acetyl group and the easy accessibility of relatively large amounts, in comparison with the endogenous factors xcex11PI and xcex12M, with the aid of the process according to the invention recommend these compounds for pharmacological evaluation for clinical pictures characterised by tissue destruction caused by HLE.
The activity of the compounds according to the invention manifests itself, for example, in the experimental emphysema model. One hour before induction of emphysema by intratracheal administration of 0.3 mg of HLE in hamsters, 0.5 mg or 2 mg of Nxcex1-acetyl-eglin C (to 8 animals in each case) were also administered intratracheally. In the unprotected animaLs (those which had not been pretreated with Nxcex1-acetyl-eglin CO the pulmonary function tests and histological examinations carried out after two months showed severe putmonary obstructions and emphysema. In contrast, all the animals pretreated with Nxcex1-acetyl-eglin C showed normal pulmonary functions. Histological examination of the lungs showed merely mild, local emphysematic changes in two of the eight animals from the tow dose group (0.5 mg of Nxcex1-acetyl-eglin C); the other animals showed no changes, which demonstrates the protective action of intratracheally administered Nxcex1-acetyl-eglin C and at the same time its low toxicity.
The novel eglin compounds according to the invention, in particular the Nxcex1-acetyl-eglin compounds, can accordingly be used for the prophylaxis and for the therapeutic treatment of pulmonary diseases, for example pulmonary diseases caused by leucocyte elastase, such as pulmonary ephysema and ARDS (xe2x80x9cacute respiratory distress syndromexe2x80x9d) and mucoviscidosis, and furthermore in cases of septic shock and as antiphlogistics and antiinflammatories. The present invention also relates to the use of the novel eglin compounds according to the invention and of their pharmaceutically acceptable salts in the prophylactic and therapeutic treatment of the clinical pictures mentioned.
The invention also relates to pharmaceutical compositions containing at least one of the compounds according to the invention or pharmaceutically acceptable salts thereof, if appropriate together with a pharmaceutically acceptable excipient and/or auxiliaries.
These compositions can be used, in particular, for the abovementioned indications, where, for example, they are administered parenterally (such as intravenously or intrapulmonarily) or applied topically. The dosage depends, in particular, on the specific processing form and on the aim of the therapy or prophylaxis.
Administration is by intravenous injection or intrapulmonarily, by inhalation, for example using a Bird apparatus. Pharmaceutical products for parenteral administration in individual-dose form accordingly contain about 10 to 50 mg of the compounds according to the invention per dose, depending on the mode of administration. Besides the active ingredient, these pharmaceutical compositions usually also contain sodium chloride, mannitol or sorbitol, to establish isotonicity. They can be in freeze-dried or dissolved form, and solutions can advantageously contain an antibacterial preservative, for example 0.2 to 0.3% of methyl or ethyl 4-hydroxybenzoate.
A product for topical application can be in the form of an aqueous solution, lotion or jelly, an oily solution or suspension, or a fat-containing or, in particular, emulsion ointment. A product in the form of an aqueous solution is obtained, for example, by dissolving the active ingredients according to the invention, or a therapeutically acceptable salt thereof, in an aqueous buffer solution of pH 4 to 7.5 and, if desired, adding a further active ingredient, for example an antiinflammatory agent, and/or a polymeric adhesive, for example polyvinylpyrrolidone, and/or a preservative. The concentration of the active ingredient is about 0.1 to about 5 mg, preferably 0.25 to 1.0 mg, in 10 ml of a solution or 10 g of a jelly.
An oily administration form for topical application is obtained, for example, by suspending the active ingredients according to the invention, or a therapeutically acceptable salt thereof, in an oil, if appropriate with the addition of swelling agents, such as aluminium stearate, and/or surface-active agents (surfactants), the HLB value (xe2x80x9chydrophilic-lipophilic balancexe2x80x9d) of which is less than 10, such as fatty acid monoesters of polyhydric alcohols, for example glycerol monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. A fat-containing ointment is obtained, for example, by suspending the active ingredients according to the invention, or salts thereof, in a spreadable fat base, if appropriate with the addition of a surfactant with an HLB value of below 10. An emulsion ointment is obtained by triturating an aqueous solution of the active ingredients according to the invention, or of salts thereof, in a soft, spreadable fat base with the addition of a surfactant, the HLB value of which is below 10. All these topical forms of application can also contain preservatives. The concentration of the active ingredient is about 0.1 to about 5 mg, preferably 0.25 to 1.0 mg, in about 10 g of the base.
Inhalation products for the treatment of the respiratory tract by intrapumonary administration are, for example, aerosols or sprays which can distribute the pharmacological active ingredient in the form of drops of a solution or suspension. Products in which the pharmacological active ingredient is in solution contain, in addition to this ingredient, a suitable propellant, and furthermore, if necessary, an additional solvent and/or a stabiliser. Instead of the propellant gas, it is also possible to use compressed air, in which case this can be produced as required by means of a suitable compression and expansion device.
Bird respirators which have been introduced into medicine and are known are particularly suitable for the administration; a solution of the active ingredient is here introduced into the apparatus, misted with a slight increased pressure and introduced into the lung of the respirated patient.
Depending on the age, individual condition and type of disease, the dosage for a warm-blooded organism (humans or animals) weighing about 70 kg is about 10 to about 30 mg per inhalation (once or twice daily) for intrapulmonary administration, and about 10 to about 1,000 mg per day for intravenous administration, for example also by continuous infusion.
Therapeutically active sputum and plasma concentrations which can be determined by means of immunological processes, such as ELISA, are between 10 and 100 xcexcg/ml (about 1 to 10 xcexcmol/l).
The invention particularly relates to the DNA sequences which are described in the examples and code an eglin or modified eglin, expression plasmids containing such DNA sequences, microorganisms transformed with such expression plasmids, monoclonal antibodies against eglins, hybridoma cells which produce such antibodies, and test kits for immunoassay containing such antibodies, the processes described in the examples for their preparation and the process described in the examples for the preparation of eglins with the aid of the transformed microorganisms, and the novel eglin compounds mentioned in the examples.
Some embodiments of the present invention which are described in the following experimental section are illustrated with the aid of the accompanying drawings.